Unraveling the mechanisms regulating the development and renewal of normal tissues is not only one of the main goals of developmental biology but also an essential step for the elucidation of the mechanisms underlying the development of pathological processes. The way the urothelium is formed and maintained is largely unknown and the existence of urothelial stem cells is controversial. This proposal aims at defining cell lineages and differentiation programs in the bladder urothelium. We have recently demonstrated that the p53-homologue p63 is required for the development of basal and intermediate cells of the urothelium. Additionally, by performing blastocyst complementation experiments we have shown that umbrella cells of the urothelium of can develop and be maintained independently from p63-positive basal and intermediate cells. These results challenge the current view that basal/intermediate cells are the progenitors of umbrella cells and support the hypothesis that basal/intermediate cells and umbrella cells represent entirely independent cell lineages. The goal of this proposal is to provide a definitive answer on whether or not this hypothesis is correct. In fact, although p63-positive basal/intermediate cells are not required for the formation of umbrella cells, it is possible that p63-positive basal/intermediate cells exhibit stem cells capabilities in other circumstances (i.e. during aging or after cell damage) or in conjunction with other cell types. Here we propose to test the central hypothesis by 1) determining if p63-positive basal and intermediate cells are required for the renewal of urothelial umbrella cells during aging or after cell injury 2) determining if p63-positive basal and intermediate cells give rise to umbrella cells by performing genetic lineage tracing. These studies will clarify lineage relationships among bladder epithelial cells and provide important insights into the nature of urothelial stem cells.